Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene. D Del Bufalo, … , A Sacchi, G Zupi J Clin Invest. 1996;98(5):1165-1173. https://doi.org/10.1172/JCI118900. Research Article Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentation. Moreover, we find that apoptosis is preceded by accumulation of the cells in the G0/G1 phase of the cell cycle. Interestingly, lonidamine fails to activate the apoptotic program in the corresponding sensitive parental cell lines (ADR-sensitive MCF-7 WT, and nitrosourea-sensitive LI cells) even after long exposure times. The evaluation of bcl-2 protein expression suggests that this different effect of lonidamine treatment in drug-resistant and -sensitive cell lines might not simply be due to dissimilar expression levels of bcl-2 protein. To determine whether the lonidamine-induced apoptosis is mediated by p53 protein, we used cells lacking endogenous p53 and overexpressing either wild-type p53 or dominant-negative p53 mutant. We find that apoptosis by lonidamine is independent of the p53 gene. Find the latest version: https://jci.me/118900/pdf Lonidamine Induces Apoptosis in Drug-resistant Cells Independently of the p53 Gene Donatella Del Bufalo,* Annamaria Biroccio,* Silvia Soddu,‡ Nina Laudonio,* Carmen D’Angelo,* Ada Sacchi,‡ and Gabriella Zupi* *Experimental Chemotherapy Laboratory and ‡Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, C.R.S., 00158 Rome, Italy Abstract indazole-3-carboxylic acid, has been shown to revert the resis- tance of tumor cells to adriamycin (ADR) and cis-platinum Lonidamine, a dichlorinated derivative of indazole-3-car- (4–6). Clinical trials performed in patients with advanced boxylic acid, was shown to play a significant role in revers- breast cancer have also demonstrated that LND significantly ing or overcoming multidrug resistance. Here, we show that increases the activity of epirubicin (7). LND decreases cell me- exposure to 50 ␮g/ml of lonidamine induces apoptosis in tabolism by reducing ATP production, and alters the cell adriamycin and nitrosourea-resistant cells (MCF-7 ADRR membrane through its affinity for the inner leaflet of the lipid human breast cancer cell line, and LB9 glioblastoma multi- bilayer (8, 9). These effects are thought to impair tumor cell form cell line), as demonstrated by sub-G1 peaks in DNA metabolic adaptations, such as reduction of drug efflux and content histograms, condensation of nuclear chromatin, detoxification, necessary for drug resistance. Indeed, we previ- and internucleosomal DNA fragmentation. Moreover, we ously showed that LND overcomes ADR resistance in the hu- find that apoptosis is preceded by accumulation of the cells man breast cancer cell line MCF-7 ADRR by increasing intra- in the G0/G1 phase of the cell cycle. Interestingly, lonidamine cellular content of the chemotherapeutic agent (4). However, fails to activate the apoptotic program in the corresponding comparing the effects of LND on the cell survival of ADR-resis- sensitive parental cell lines (ADR-sensitive MCF-7 WT, and tant and -sensitive cells, we found that with similar intracellu- nitrosourea-sensitive LI cells) even after long exposure times. lar ADR levels, MCF-7 ADRR cells were much more affected The evaluation of bcl-2 protein expression suggests that this than MCF-7 WT cells (4). Moreover, the intracellular accumu- different effect of lonidamine treatment in drug-resistant lation of ADR induced by LND remained localized in the cy- and -sensitive cell lines might not simply be due to dissimi- toplasm (10). Thus, modification of intracellular levels of ADR lar expression levels of bcl-2 protein. alone is not sufficient to explain the drastic reversal of ADR- To determine whether the lonidamine-induced apoptosis resistant phenotype induced by LND. is mediated by p53 protein, we used cells lacking endoge- It has recently been shown that a variety of currently used nous p53 and overexpressing either wild-type p53 or domi- cancer chemotherapeutic agents exert their effects by inducing nant-negative p53 mutant. We find that apoptosis by lonida- apoptotic cell death (11–13). Indeed, the ability of tumor cells mine is independent of the p53 gene. (J. Clin. Invest. 1996. to respond to damage and eventually activate the apoptotic 98:1165–1173.) Key words: chemoresistance • breast cancer program might determine the ultimate success of cancer ther- • glioblastoma • bcl-2 • DNA fragmentation apy (14). Apoptosis appears to play a pivotal role in the con- trol of tumor growth by counterbalancing proliferation (15– Introduction 17). Genes involved in neoplastic transformation, such as p53 and bcl-2 are also involved in apoptosis regulation (18), with Drug resistance of tumor cells is one of the major obstacles to the p53 gene being the most frequently altered gene in human effective chemotherapy (1). The aim of numerous recent stud- cancers (19). Wild-type p53 (wt-p53) protein has been shown ies has been to identify agents capable of reversing this resis- to be a mediator of different types of apoptosis, including that 1 tance (2, 3). Lonidamine (LND), a dichlorinated derivative of induced by several chemotherapeutic agents such as adriamy- cin, etoposide, and 5-fluorouracil (20). More interestingly, mu- tations or deletions in the p53 gene have been associated, in vivo and in vitro, with resistance to apoptosis induced by the Address correspondence to Gabriella Zupi, Experimental Chemo- same chemotherapeutic agents (14). The bcl-2 gene is able to therapy Laboratory, Regina Elena Cancer Institute, C.R.S. Via delle confer resistance to apoptosis in several systems (21, 22), and it Messi d’Oro 156, 00158 Rome, Italy. Phone: 39-6-49852537; FAX: 39- was found to be overexpressed in follicular lymphomas and 6-49852505. leukemias (23, 24). Moreover, expression of bcl-2 is one poten- Received for publication 23 April 1996 and accepted in revised tial mechanism by which tumor cells escape p53-mediated apop- form 25 June 1996. tosis (25). Here, we report the results obtained by studying the mech- 1. Abbreviations used in this paper: ADR, adriamycin; BCNU, 1,3- anism(s) through which LND rescues the resistant phenotype bis(2-chloroethyl)-1-nitrosourea; LND, lonidamine; PI, propidium io- dide; ts-p53, temperature sensitive p53; vinc, vincristine; wt-p53, wild- of tumor cells. We used two drug-resistant cell lines which dif- type p53. fer in histotype and mechanism of drug resistance: ADR-resis- tant breast cancer cells (MCF-7 ADRR) and nitrosourea-resis- J. Clin. Invest. tant glioblastoma cells (LB9). We found that LND induces © The American Society for Clinical Investigation, Inc. apoptosis in the resistant cells, whereas it does not in the pa- 0021-9738/96/09/1165/09 $2.00 rental-sensitive cells (MCF-7 WT, and LI). In addition, over- Volume 98, Number 5, September 1996, 1165–1173 expression of mutant-p53 or wt-p53 proteins as well as the use p53-independent LND-induced Apoptosis in Drug-resistant Cancer Lines 1165 of the p53-negative promyelocytic leukemia cell line resistant from the plates by trypsin-EDTA treatment. Cells were washed, as- to vincristine (HL60/Vinc) (26), showed that LND-induced ap- sayed for cell viability (trypan blue exclusion test), and counted (ZM optosis is not mediated by p53. These results suggest that LND Coulter Counter; Kontron Instruments, Milan, Italy). Aliquots of might be useful in the treatment of drug-resistant tumors pre- LND-treated and control cells were differentially processed accord- senting p53 alterations (27, 28). ing to the analyses to be performed. Flow cytometry. LND-induced apoptosis was detected by flow cytometric analysis of permeabilized, propidium iodide (PI)–stained cells according to the method of Telford et al. (31). Samples (106 Methods cells) were washed once in PBS and the pellets resuspended in 80% ethanol at 4ЊC for at least 60 min. For detection of apoptosis, fixed Cell lines and culture conditions. The breast cancer cell line MCF-7 cells were centrifuged, resuspended in PBS (4 ϫ 106 cell/ml), and WT and its ADR-resistant derivative, MCF-7 ADRR, were kindly maintained at 37ЊC for 20 min before staining with a 2ϫ solution of provided by Dr. K. Cowan (National Cancer Institute, Bethesda, 0.1% Triton X-100, 0.1 mM EDTA(Na)2, 50 ␮g/ml RNase A, and 50 MD). The LI line was established from surgical specimens of a glio- ␮g/ml PI in PBS. Samples were stored in the dark at room tempera- blastoma multiform (29), and its 1,3-bis(2-chloroethyl)-1-nitrosourea ture and analyzed with a FACScan® (Becton Dickinson & Co., San (BCNU)–resistant derivative (LB9) was kindly provided by Dr. A. Jose, CA). For cell cycle analysis, 2 ϫ 105 fixed cells were centrifuged, Floridi (Regina Elena Cancer Institute, Rome, Italy). The vincristine- resuspended in a solution containing 750 ␮g/ml RNase A, and 50 ␮g/ml resistant derivative HL60 promyelocytic leukemia cell line (HL60/ of PI. At least 2 ϫ 104 events/sample were acquired in list mode by a Vinc) was kindly provided by Dr. C. Cucco (Regina Elena Cancer In- Consort 32 minicomputer (Hewlett-Packard Co., Palo Alto, CA). stitute) (30). The tumor lines were maintained in 25 cm2 tissue culture Cell percentages in the different phases of the cell cycle were esti- flasks in RPMI 1640 medium supplemented with 10% fetal calf se- mated according to Fox’s method (32). rum, 2 mM l-glutamine, and antibiotics in a humidified atmosphere Morphological examination.